The aim of this proposed research is to discern the role of putative protein domains encoded by plant disease-resistance (R) genes. These putative functional domains are predicted to be involved in protein:protein interactions, nucleotide binding, and signal transduction, based on sequence comparisons with well-characterized proteins. The tobacco N gene, which confers resistance to tobacco mosaic virus (TMV), will be the R gene utilized in these studies. Numerous biochemical methods will be employed to disect the function of N protein domains in TMV recognition and signal transduction. The specific aims of this proposal are (1) to determine the relative levels of full-length and truncated (Ntr) protein levels in TMV infected and uninfected tobacco using immunoblot analysis, (2) to overexpress N and TMV replicase proteins in E.coli and insect cells for in vitro biochemical analysis of activity, protein-protein interactions, structural studies, and antibody production, (3) to determine the biochemical properties of N including nucleotide binding and hydrolysis, and phosphorylation, (4) to test for in vivo and in vitro interactions between N and TMV replicase proteins using co-immunoprecipitation and affinity chromatography, and (5) to identify N-interacting proteins using co-purification, affinity chromatography, and interaction cloning. These biochemical approaches will complement genetic and molecular techniques currently being applied in the Baker lab to understand N function. A detailed understanding of N function will contribute to engineering of novel, disease-resistant crops, which will increase crop yields while reducing the use of chemical pesticides. In addition, similarities between N and the mammalian interleukin-1 receptor suggest that characterization of N may help to understand the signaling mechanisms of the interleukin-1 receptor-mediated pathway. Defects in the latter contribute to the pathogenesis of certain cancers and AIDS in humans.